Research Article: Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11

Date Published: March 22, 2016

Publisher: Public Library of Science

Author(s): Igor Minia, Christine Clayton, David Horn.


The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2), and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3′-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress.

Partial Text

The African trypanosome Trypanosoma brucei is responsible for sleeping sickness in humans and nagana in livestock. Bloodstream-form trypanosomes, which are found in mammalian blood and tissue fluids, are exposed to temperatures ranging from about 36°C to 40°C (fever). Trypanosomes are transmitted by Tsetse flies, where they replicate as procyclic forms in the midgut, progressing to epimastigotes, then metacyclic forms in the salivary glands. Within Tsetse, the temperature may fluctuate between 20°C and 43°C [1]. In addition, availability of nutrients in the two hosts is different. Trypanosomes, like other kinetoplastids, manage these changes almost exclusively through post-transcriptional mechanisms. Transcription is polycistronic and individual mRNAs are generated by processing: this precludes transcription control at the level of individual open reading frames. In contrast, there is extensive evidence for regulation of mRNA stability [2] and translation [3,4]. This regulation is often determined by sequences in the 3′-untranslated regions (3′-UTRs) of mRNAs, and mediated by RNA binding proteins [5–8].

The amount of ZC3H11 was increased by stresses that cause the accumulation either of protein fragments, or of incompletely folded proteins in the cytosol. ZC3H11 protein abundance was, in contrast, not affected by inducers of ER stress. This result corresponds to the role of ZC3H11 in stabilizing mRNAs that encode proteins involved in refolding cytosolic proteins [20]. The pattern of induction suggests that the existence of (partially) unfolded proteins might be the stimulus that causes ZC3H11 accumulation. However, there is a conundrum, since ZC3H11 expression is increased at 37°C in procyclic forms, but repressed at the same temperature in bloodstream forms. Putting procyclic forms at 37°C is most unlikely to cause mass protein denaturation, since the bulk of the procyclic proteome is identical to that of bloodstream forms. It is however possible that some procyclic-specific proteins are heat-sensitive.




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